Search

The purpose of this study was to investigate the rear knee angle range in the set position that allows sprinters to reach greater propulsion on the rear block during the sprint start. Eleven university-track team sprinters performed the sprint start using three rear knee angle conditions: 90°, 115° and 135°. A motion capture system consisting of 8 digital cameras (250 Hz) was used to record kinematic parameters at the starting block phase and the acceleration phase. The following variables were considered: horizontal velocity of the centre of mass (COM), COM height, block time, pushing time on the rear block, percentage of pushing time on the rear block, force impulse, push-off angle and length of the first two strides. The main results show that first, horizontal block velocity is significantly greater at 90° vs 115° and 135° rear knee angle (p

Chitosan-based nanoparticles (chiNPs) are considered to be potentially good carriers for the sustained intracellular delivery of specific molecules. However, scarce attention has been paid to the long-lasting permanence of these NPs in the intracellular milieu, as well as to their intracellular fate (i.e., distribution, interaction with cell organelles, and degradation) in the long term. In the present study, the presence and subcellular location of FITC-labelled chiNPs were monitored in HeLa cells up to 14 days post-administration using multicolor-fluorescence confocal microscopy and diaminobenzidine photo-oxidation at transmission electron microscopy. The main result of the present study is the demonstration that internalized chiNPs persist inside the cell up to two weeks, occurring in both the cytoplasm and nucleus; accordingly, chiNPs are able to pass from mother to daughter cells through several mitotic cycles. The cells did not show increased mortality or structural damage up to 14 days after chiNP exposure.

Diaminobenzidine photoconversion is a technique by which a fluorescent dye is transformed into a stably insoluble, brown, electrondense signal, thus enabling examination at both bright field light microscopy and transmission electron microscopy. In this work, a procedure is proposed for combining photoconversion and immunoelectron microscopy: in vitro cell cultures have been first submitted to photoconversion to analyse the intracellular fate of either fluorescent nanoparticles or photosensitizing molecules, then processed for transmission electron microscopy; different fixative solutions and embedding media have been used, and the ultrathin sections were finally submitted to post-embedding immunogold cytochemistry. Under all conditions the photoconversion reaction product and the target antigen were properly detected in the same section; Epon-embedded, osmicated samples required a pre-treatment with sodium metaperiodate to unmask the antigenic sites. This simple and reliable procedure exploits a single sample to simultaneously localise the photoconversion product and a variety of antigens allowing a specific identification of subcellular organelles at the ultrastructural level.

Chitosan-based nanoparticles (NPs) deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier) and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB) photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus.

To realize the benefits of a hydrogen economy, hydrogen must be produced cleanly, efficiently and affordably from renewable resources and, preferentially, close to the end-users. The goal is a sustainable cycle of hydrogen production and use: in the first stage of the cycle, hydrogen is produced from renewable resources and then used to feed a fuel cell. This cycle produces no pollution and no greenhouse gases. In this context, the development of electrolyzers producing high-purity hydrogen with a high efficiency and low cost is of great importance. Electrode materials play a fundamental role in influencing electrolyzer performances; consequently, in recent years considerable efforts have been made to obtain highly efficient and inexpensive catalyst materials. To reach both goals, we have developed electrodes based on Pd-Co alloys to be potentially used in the PEMEL electrolyzer. In fact, the Pd-Co alloy is a valid alternative to Pt for hydrogen evolution. The alloys were electrodeposited using two different types of support: carbon paper, to fabricate a porous structure, and anodic alumina membrane, to obtain regular arrays of nanowires. The goal was to obtain electrodes with very large active surface areas and a small amount of material. The research demonstrates that the electrochemical method is an ideal technique to obtain materials with good performances for the hydrogen evolution reaction. The Pd-Co alloy composition can be controlled by adjusting electrodeposition parameters (bath composition, current density and deposition time). The main results concerning the fabrication process and the characterization are presented and the performance in acid conditions is discussed.

Calcium phosphate/chitosan/collagen composite coating on AISI 304 stainless steel was investigated. Coatings were realized by galvanic coupling that occurs without an external power supply because it begins with the coupling between two metals with different standard electrochemical potentials. The process consists of the co-deposition of the three components with the calcium phosphate crystals incorporated into the polymeric composite of chitosan and collagen. Physical-chemical characterizations of the samples were executed to evaluate morphology and chemical composition. Morphological analyses have shown that the surface of the stainless steel is covered by the deposit, which has a very rough surface. XRD, Raman, and FTIR characterizations highlighted the presence of both calcium phosphate compounds and polymers. The coatings undergo a profound variation after aging in simulated body fluid, both in terms of composition and structure. The tests, carried out in simulated body fluid to scrutinize the corrosion resistance, have shown the protective behavior of the coating. In particular, the corrosion potential moved toward higher values with respect to uncoated steel, while the corrosion current density decreased. This good behavior was further confirmed by the very low quantification of the metal ions (practically absent) released in simulated body fluid during aging. Cytotoxicity tests using a pre-osteoblasts MC3T3-E1 cell line were also performed that attest the biocompatibility of the coating.

Wearable sensors for sweat biomarkers can provide facile analyte capability and monitoring for several diseases. In this work, a green wearable sensor for sweat absorption and chloride sensing is presented. In order to produce a sustainable device, polylactic acid (PLA) was used for both the substrate and the sweat absorption pad fabrication. The sensor material for chloride detection consisted of silver-based reference, working, and counter electrodes obtained from upcycled compact discs. The PLA substrates were prepared by thermal bonding of PLA sheets obtained via a flat die extruder, prototyped in single functional layers via CO 2 laser cutting, and bonded via hot-press. The effect of cold plasma treatment on the transparency and bonding strength of PLA sheets was investigated. The PLA membrane, to act as a sweat absorption pad, was directly deposited onto the membrane holder layer by means of an electrolyte-assisted electrospinning technique. The membrane adhesion capacity was investigated by indentation tests in both dry and wet modes. The integrated device made of PLA and silver-based electrodes was used to quantify chloride ions. The calibration tests revealed that the proposed sensor platform could quantify chloride ions in a sensitive and reproducible way. The chloride ions were also quantified in a real sweat sample collected from a healthy volunteer. Therefore, we demonstrated the feasibility of a green and integrated sweat sensor that can be applied directly on human skin to quantify chloride ions.

Quantification of oxidative stress is a challenging task that can help in monitoring chronic inflammatory respiratory airway diseases. Different studies can be found in the literature regarding the development of electrochemical sensors for H 2 O 2 in cell culture medium to quantify oxidative stress. However, there are very limited data regarding the impact of the cell culture medium on the electrochemical quantification of H 2 O 2 . In this work, we studied the effect of different media (RPMI, MEM, DMEM, Ham's F12 and BEGM/DMEM) on the electrochemical quantification of H 2 O 2 . The used electrode is based on reduced graphene oxide (rGO) and gold nanoparticles (AuNPs) and was obtained by co-electrodeposition. To reduce the electrode fouling by the medium, the effect of dilution was investigated using diluted (50% v / v in PBS) and undiluted media. With the same aim, two electrochemical techniques were employed, chronoamperometry (CH) and linear scan voltammetry (LSV). The influence of different interfering species and the effect of the operating temperature of 37 °C were also studied in order to simulate the operation of the sensor in the culture plate. The LSV technique made the sensor adaptable to undiluted media because the test time is short, compared with the CH technique, reducing the electrode fouling. The long-term stability of the sensors was also evaluated by testing different storage conditions. By storing the electrode at 4 °C, the sensor performance was not reduced for up to 21 days. The sensors were validated measuring H 2 O 2 released by two different human bronchial epithelial cell lines (A549, 16HBE) and human primary bronchial epithelial cells (PBEC) grown in RPMI, MEM and BEGM/DMEM media. To confirm the results obtained with the sensor, the release of reactive oxygen species was also evaluated with a standard flow cytometry technique. The results obtained with the two techniques were very similar. Thus, the LSV technique permits using the proposed sensor for an effective oxidative stress quantification in different culture media and without dilution.

In this work, composite coatings of chitosan and silver nanoparticles were presented as an antibacterial coating for orthopedic implants. Coatings were deposited on AISI 304L using the galvanic deposition method. In galvanic deposition, the difference of the electrochemical redox potential between two metals (the substrate and a sacrificial anode) has the pivotal role in the process. In the coupling of these two metals a spontaneous redox reaction occurs and thus no external power supply is necessary. Using this process, a uniform deposition on the exposed area and a good adherence of the composite coating on the metallic substrate were achieved. Physical-chemical characterizations were carried out to evaluate morphology, chemical composition, and the presence of silver nanoparticles. These characterizations have shown the deposition of coatings with homogenous and porous surface structures with silver nanoparticles incorporated and distributed into the polymeric matrix. Corrosion tests were also carried out in a simulated body fluid at 37 °C in order to simulate the same physiological conditions. Corrosion potential and corrosion current density were obtained from the polarization curves by Tafel extrapolation. The results show an improvement in protection against corrosion phenomena compared to bare AISI 304L. Furthermore, the ability of the coating to release the Ag + was evaluated in the simulated body fluid at 37 °C and it was found that the release mechanism switches from anomalous to diffusion controlled after 3 h.

Hybrid porous scaffolds composed of both natural and synthetic biopolymers have demonstrated significant improvements in the tissue engineering field. This study investigates for the first time the fabrication route and characterization of poly-L-lactic acid scaffolds blended with polyhydroxyalkanoate up to 30 wt%. The hybrid scaffolds were prepared by a thermally induced phase separation method starting from ternary solutions. The microstructure of the hybrid porous structures was analyzed by scanning electron microscopy and related to the blend composition. The porosity and the wettability of the scaffolds were evaluated through gravimetric and water contact angle measurements, respectively. The scaffolds were also characterized in terms of the surface chemical properties via Fourier transform infrared spectroscopy in attenuated total reflectance. The mechanical properties were analyzed through tensile tests, while the crystallinity of the PLLA/PHA scaffolds was investigated by differential scanning calorimetry and X-ray diffraction.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer. This article is highlighted in the In This Issue feature, p. 2659 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

Advances in DNA sequencing technologies have reshaped our understanding of the molecular basis of cancer, providing a precise genomic view of tumors. Complementary biochemical and biophysical perspectives of cancer point toward profound shifts in nutrient uptake and utilization that propel tumor growth and major changes in the structure of the plasma membrane of tumor cells. The molecular mechanisms that bridge these fundamental aspects of tumor biology remain poorly understood. Here, we show that the lysophosphatidylcholine acyltransferase LPCAT1 functionally links specific genetic alterations in cancer with aberrant metabolism and plasma membrane remodeling to drive tumor growth. Growth factor receptor-driven cancers are found to depend on LPCAT1 to shape plasma membrane composition through enhanced saturated phosphatidylcholine content that is, in turn, required for the transduction of oncogenic signals. These results point to a genotype-informed strategy that prioritizes lipid remodeling pathways as therapeutic targets for diverse cancers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Precision oncology hinges on linking tumour genotype with molecularly targeted drugs 1 ; however, targeting the frequently dysregulated metabolic landscape of cancer has proven to be a major challenge 2 . Here we show that tissue context is the major determinant of dependence on the nicotinamide adenine dinucleotide (NAD) metabolic pathway in cancer. By analysing more than 7,000 tumours and 2,600 matched normal samples of 19 tissue types, coupled with mathematical modelling and extensive in vitro and in vivo analyses, we identify a simple and actionable set of 'rules'. If the rate-limiting enzyme of de novo NAD synthesis, NAPRT, is highly expressed in a normal tissue type, cancers that arise from that tissue will have a high frequency of NAPRT amplification and be completely and irreversibly dependent on NAPRT for survival. By contrast, tumours that arise from normal tissues that do not express NAPRT highly are entirely dependent on the NAD salvage pathway for survival. We identify the previously unknown enhancer that underlies this dependence. Amplification of NAPRT is shown to generate a pharmacologically actionable tumour cell dependence for survival. Dependence on another rate-limiting enzyme of the NAD synthesis pathway, NAMPT, as a result of enhancer remodelling is subject to resistance by NMRK1-dependent synthesis of NAD. These results identify a central role for tissue context in determining the choice of NAD biosynthetic pathway, explain the failure of NAMPT inhibitors, and pave the way for more effective treatments.

We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFR A289D/T/V ). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFR A289D/T/V mutants, corroborated in mice bearing intracranial tumors expressing EGFR A289V and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFR A289V tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFR A289V mutation in glioblastoma, postulating EGFR A289V as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This corrects the article DOI: 10.1038/ncomms15223.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity., (© 2017 Zanca et al.; Published by Cold Spring Harbor Laboratory Press.)

This corrects the article DOI: 10.1038/cdd.2010.65.

Glioblastoma (GBM) is the most lethal type of human brain cancer, where deletions and mutations in the tumour suppressor gene PTEN (phosphatase and tensin homolog) are frequent events and are associated with therapeutic resistance. Herein, we report a novel chromatin-associated function of PTEN in complex with the histone chaperone DAXX and the histone variant H3.3. We show that PTEN interacts with DAXX and, in turn PTEN directly regulates oncogene expression by modulating DAXX-H3.3 association on the chromatin, independently of PTEN enzymatic activity. Furthermore, DAXX inhibition specifically suppresses tumour growth and improves the survival of orthotopically engrafted mice implanted with human PTEN-deficient glioma samples, associated with global H3.3 genomic distribution changes leading to upregulation of tumour suppressor genes and downregulation of oncogenes. Moreover, DAXX expression anti-correlates with PTEN expression in GBM patient samples. Since loss of chromosome 10 and PTEN are common events in cancer, this synthetic growth defect mediated by DAXX suppression represents a therapeutic opportunity to inhibit tumorigenesis specifically in the context of PTEN deletion.

Small-molecule inhibitors targeting growth factor receptors have failed to show efficacy for brain cancers, potentially due to their inability to achieve sufficient drug levels in the CNS. Targeting non-oncogene tumor co-dependencies provides an alternative approach, particularly if drugs with high brain penetration can be identified. Here we demonstrate that the highly lethal brain cancer glioblastoma (GBM) is remarkably dependent on cholesterol for survival, rendering these tumors sensitive to Liver X receptor (LXR) agonist-dependent cell death. We show that LXR-623, a clinically viable, highly brain-penetrant LXRα-partial/LXRβ-full agonist selectively kills GBM cells in an LXRβ- and cholesterol-dependent fashion, causing tumor regression and prolonged survival in mouse models. Thus, a metabolic co-dependency provides a pharmacological means to kill growth factor-activated cancers in the CNS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

With the evolution of technology, there is now a deeper understanding of glioblastoma as an inter- and intraheterogeneous disease comprising a multitude of genetically and epigenetically different cancer cells. Greater characterization of glioblastoma at the molecular level has improved its initial pathophysiological staging and classification. With this knowledge comes the hope that more efficacious therapies to combat this highly lethal disease are on the horizon. One possibility for intervention is represented by the targeting of epidermal growth factor receptor (EGFR), which is amplified and mutated in a large subset of patients. In this review, we provide a brief overview of EGFR and its mutated form, EGFR variant III, describing the downstream cellular pathways activated by each receptor, available animal models, therapeutic strategies to inhibit the receptor, and possible intervention routes to efficiently target this receptor and prevent the emergence of resistant mechanisms which to date have hampered a successful therapeutic outcome., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCβ mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCβ is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

PED/PEA-15 is a death effector domain (DED) family member with a variety of effects on cell growth and metabolism. To get further insight into the role of PED in cancer, we aimed to find new PED interactors. Using tandem affinity purification, we identified HSC70 (Heat Shock Cognate Protein of 70 kDa)-which, among other processes, is involved in chaperone-mediated autophagy (CMA)-as a PED-interacting protein. We found that PED has two CMA-like motifs (i.e., KFERQ), one of which is located within a phosphorylation site, and demonstrate that PED is a bona fide CMA substrate and the first example in which phosphorylation modifies the ability of HSC70 to access KFERQ-like motifs and target the protein for lysosomal degradation. Phosphorylation of PED switches its function from tumor suppression to tumor promotion, and we show that HSC70 preferentially targets the unphosphorylated form of PED to CMA. Therefore, we propose that the up-regulated CMA activity characteristic of most types of cancer cell enhances oncogenesis by shifting the balance of PED function toward tumor promotion. This mechanism is consistent with the notion of a therapeutic potential for targeting CMA in cancer, as inhibition of this autophagic pathway may help restore a physiological ratio of PED forms., (© 2014 Wiley Periodicals, Inc.)

The EGF receptor (EGFR) is amplified and mutated in glioblastoma, in which its common mutation (ΔEGFR, also called EGFRvIII) has a variety of activities that promote growth and inhibit death, thereby conferring a strong tumor-enhancing effect. This range of activities suggested to us that ΔEGFR might exert its influence through pleiotropic effectors, and we hypothesized that microRNAs might serve such a function. Here, we report that ΔEGFR specifically suppresses one such microRNA, namely miR-9, through the Ras/PI3K/AKT axis that it is known to activate. Correspondingly, expression of miR-9 antagonizes the tumor growth advantage conferred by ΔEGFR. Silencing of FOXP1, a miR-9 target, inhibits ΔEGFR-dependent tumor growth and, conversely, de-repression of FOXP1, as a consequence of miR-9 inhibition, increases tumorigenicity. FOXP1 was sufficient to increase tumor growth in the absence of oncogenic ΔEGFR signaling. The significance of these findings is underscored by our finding that high FOXP1 expression predicts poor survival in a cohort of 131 patients with glioblastoma. Collectively, these data suggest a novel regulatory mechanism by which ΔEGFR suppression of miR-9 upregulates FOXP1 to increase tumorigenicity., (©2014 AACR)

Receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) regulate cellular homeostatic processes. EGFR activates downstream signaling cascades that promote tumor cell survival, proliferation and migration. Dysregulation of EGFR signaling as a consequence of overexpression, amplification and mutation of the EGFR gene occurs frequently in several types of cancers and many become dependent on EGFR signaling to maintain their malignant phenotypes. Consequently, concerted efforts have been mounted to develop therapeutic agents and strategies to effectively inhibit EGFR. However, limited therapeutic benefits to cancer patients have been derived from EGFR-targeted therapies. A well-documented obstacle to improved patient survival is the presence of EGFR-inhibitor resistant tumor cell variants within heterogeneous tumor cell masses. Here, we summarize the mechanisms by which tumors resist EGFR-targeted therapies and highlight the emerging role of microRNAs (miRs) as downstream effector molecules utilized by EGFR to promote tumor initiation, progression and that play a role in resistance to EGFR inhibitors. We also examine evidence supporting the utility of miRs as predictors of response to targeted therapies and novel therapeutic agents to circumvent EGFR-inhibitor resistance mechanisms.

Purpose: mTOR pathway hyperactivation occurs in approximately 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here, we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacologic strategy to overcome it., Experimental Design: We conducted in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin-resistant signaling and blocking glioblastoma growth and a novel single-cell technology-DNA Encoded Antibody Libraries-was used to identify mechanisms of resistance., Results: Here, we show that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling, block mTORC2 signaling, and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes glioblastoma cells and orthotopic xenografts to CC214-1- and CC214-2-induced cell death., Conclusions: These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in glioblastoma, and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. In addition, this study also shows a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacologic strategy for overcoming it., (©2013 AACR.)

Chemotherapy and molecularly targeted approaches represent two very different modes of cancer treatment and each is associated with unique benefits and limitations. Both types of therapy share the overarching limitation of the emergence of drug resistance, which prevents these drugs from eliciting lasting clinical benefit. This review will provide an overview of the various mechanisms of resistance to each of these classes of drugs and examples of drug combinations that have been tested clinically. This analysis supports the contention that understanding modes of resistance to both chemotherapy and molecularly targeted therapies may be very useful in selecting those drugs of each class that will have complementing mechanisms of sensitivity and thereby represent reasonable combination therapies.

Despite their nearly universal activation of mammalian target of rapamycin (mTOR) signaling, glioblastomas (GBMs) are strikingly resistant to mTOR-targeted therapy. We analyzed GBM cell lines, patient-derived tumor cell cultures, and clinical samples from patients in phase 1 clinical trials, and find that the promyelocytic leukemia (PML) gene mediates resistance to mTOR-targeted therapies. Direct mTOR inhibitors and EGF receptor (EGFR) inhibitors that block downstream mTOR signaling promote nuclear PML expression in GBMs, and genetic overexpression and knockdown approaches demonstrate that PML prevents mTOR and EGFR inhibitor-dependent cell death. Low doses of the PML inhibitor, arsenic trioxide, abrogate PML expression and reverse mTOR kinase inhibitor resistance in vivo, thus markedly inhibiting tumor growth and promoting tumor cell death in mice. These results identify a unique role for PML in mTOR and EGFR inhibitor resistance and provide a strong rationale for a combination therapeutic strategy to overcome it.

MicroRNAs (miRNAs) have an important role in the development of chemosensitivity or chemoresistance in different types of cancer. Activation of the ERK1/2 pathway is a major determinant of diverse cellular processes and cancer development and is responsible for the transcription of several important miRNAs. Here we show a link between the ERK1/2 pathway and BIM expression through miR-494. We blocked ERK1/2 nuclear activity through the overexpression of an ERK1/2 natural interactor, the protein PED/PEA15, and we performed a microRNA expression profile. miR-494 was the most down-regulated microRNA after ERK1/2 inactivation. Moreover, we found that miR-494 induced Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance in non-small-cell lung cancer (NSCLC) through the down-modulation of BIM. Elucidation of this undiscovered ERK1/2 pathway that regulates apoptosis and cell proliferation through miR-494 in NSCLC will greatly enhance our understanding of the mechanisms responsible for TRAIL resistance and will provide an additional arm for the development of anticancer therapies.

Glioblastoma is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, survival of patients remains poor. In order to identify microRNAs (miRs) involved in glioma tumorigenesis, we evaluated, by a miRarray, differential expression of miRs in the tumorigenic glioma LN-18, LN-229 and U87MG cells compared with the non-tumorigenic T98G cells. Among different miRs we focused our attention on miR-221 and -222. We demonstrated the presence of a binding site for these two miRs in the 3' untranslated region of the protein tyrosine phosphatase μ (PTPμ). Previous studies indicated that PTPμ suppresses cell migration and is downregulated in glioblastoma. Significantly, we found that miR-221 and -222 overexpression induced a downregulation of PTPμ as analyzed by both western blot and real-time PCR. Furthermore, miR-222 and -221 induced an increase in cell migration and growth in soft agar in glioma cells. Interestingly, the re-expression of PTPμ gene was able to revert the miR-222 and -221 effects on cell migration. Furthermore, we found an inverse correlation between miR-221 and -222 and PTPμ in human glioma cancer samples. In conclusion, our results suggest that miR-221 and -222 regulate glioma tumorigenesis at least in part through the control of PTPμ protein expression.

Akt is a serine-threonine kinase that has an important role in transducing survival signals. Akt also regulates a number of proteins involved in the apoptotic process. To find new Akt interactors, we performed a two-hybrid screening in yeast using full-length Akt cDNA as bait and a human cDNA heart library as prey. Among 200 clones obtained, two of them were identified as coding for the c-FLIP(L) protein. c-FLIP(L) is an endogenous inhibitor of death receptor-induced apoptosis through the caspase-8 pathway. Using co-immunoprecipitation experiments of either transfected or endogenous proteins, we confirmed the interaction between Akt and c-FLIP(L). Furthermore, we observed that c-FLIP(L) overexpression interferes with Gsk3-β phosphorylation levels. Moreover, through its effects on Gsk3β, c-FLIP(L) overexpression in cancer cells induced resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This effect was mediated by the regulation of p27(Kip1) and caspase-3 expression. These results indicate the existence of a new mechanism of resistance to TRAIL in cancer cells, and unexpected functions of c-FLIP(L).

PED (phosphoprotein enriched in diabetes) is a 15 kDa protein involved in many cellular pathways and human diseases including type II diabetes and cancer. We recently reported that PED is overexpressed in human cancers and mediates resistance to induced apoptosis. To better understand its role in cancer, we investigated on PED interactome in non-small cell lung cancer (NSCLC). By the Tandem Affinity Purification (TAP), we identified and characterized among others, Rac1, a member of mammalian Rho GTPase protein family, as PED-interacting protein. In this study we show that PED coadiuvates Rac1 activation by regulating AKT mediated Rac1-Ser(71) phosphorylation. Furthermore, we show that the expression of a constitutively active Rac, affected PED-Ser(104) phosphorylation, which is important for PED-regulated ERK 1/2 nuclear localization. Through specific Rac1-siRNA or its pharmacological inhibition, we demonstrate that PED augments migration and invasion in a Rac1-dependent manner in NSCLC. In conclusion, we show for the first time that PED and Rac1 interact and that this interaction modulates cell migration/invasion processes in cancer cells through ERK1/2 pathway., ((c) 2010 Wiley-Liss, Inc.)

PED/PEA-15 (PED) is a death effector domain family member of 15 kDa with a broad antiapoptotic function found overexpressed in a number of different human tumors, including lung cancer. To date, the mechanisms that regulate PED expression are unknown. Therefore, we address this point by the identification of microRNAs that in non-small cell lung cancer (NSCLC) modulate PED levels. In this work, we identify miR-212 as a negative regulator of PED expression. We also show that ectopic expression of this miR increases tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death in NSCLC cells. In contrast, inhibition of endogenous miR-212 by use of antago-miR results in increase of PED protein expression and resistance to TRAIL treatment. Besides, in NSCLC, we show both in vitro and in vivo that PED and miR-212 expressions are inversely correlated, that is, PED is upregulated and miR-212 is rarely expressed. In conclusion, these findings suggest that miR-212 should be considered as a tumor suppressor because it negatively regulates the antiapoptotic protein PED and regulates TRAIL sensitivity., ((c)2010 AACR.)

In spite of growing evidence linking vitamin D(3) levels to mental health disorders, little is known about its direct targets in the brain. This study set out to investigate targets of vitamin D(3) in a human brain stem cell line. We employed arrays with antibodies directed against more than 600 structural and signalling proteins, including phospho-variants. Over 180 proteins responded to vitamin D(3), such as cyclin-dependent protein-serine kinase 1/2, epidermal growth factor receptor-tyrosine kinase, protein kinase A, protein-serine kinase Bgamma and protein-serine kinase Calpha. PEA-15 (phosphoprotein enriched in astrocytes-15 kD, also known as PED), known to be involved in various anti-proliferative and anti-apoptotic effects, was strongly up-regulated. In silico promoter analysis revealed conserved binding sites for vitamin D(3) receptor, suggesting a strong vitamin D(3) dependency of the PEA-15 promoter. PEA-15 up-regulation by vitamin D(3) could be confirmed by Western blot in two different cell lines. Analysis of mRNA and protein phosphorylation status of PEA-15 suggests that increased PEA-15 promoter activity and increased protein stabilization contribute to the overall rise of PEA-15 protein. In a functional test of this novel pathway, we demonstrated that vitamin D(3) was able to rescue cells from TRAIL-induced apoptosis through regulation of the PEA-15 expression and function. Summarized, our study presents novel targets of vitamin D(3) relevant for apoptosis and cell proliferation, and thus strongly supports a function of vitamin D(3) in the brain that impacts on processes highly relevant for major neurological disorders.

PED (phosphoprotein enriched in diabetes) is a death-effector domain (DED) family member with a broad anti-apoptotic action. PED inhibits the assembly of the death-inducing signalling complex (DISC) of death receptors following stimulation. Recently, we reported that the expression of PED is increased in breast cancer cells and determines the refractoriness of these cells to anticancer therapy. In the present study, we focused on the role of PED in non-small cell lung cancer (NSCLC), a tumour frequently characterized by evasion of apoptosis and drug resistance. Immunohistochemical analysis of a tissue microarray, containing 160 lung cancer samples, indicated that PED was strongly expressed in different lung tumour types. Western blotting performed with specimens from NSCLC-affected patients showed that PED was strongly up-regulated (>6 fold) in the areas of tumour compared to adjacent normal tissue. Furthermore, PED expression levels in NSCLC cell lines correlated with their resistance to tumour necrosis factor related apoptosis-inducing ligand (TRAIL)-induced cell death. The involvement of PED in the refractoriness to TRAIL-induced cell death was investigated by silencing PED expression in TRAIL-resistant NSCLC cells with small interfering (si) RNAs: transfection with PED siRNA, but not with cFLIP siRNA, sensitized cells to TRAIL-induced cell death. In conclusion, PED is specifically overexpressed in lung tumour tissue and contributes to TRAIL resistance.

Aims: Contrast media (CM) induce a direct toxic effect on renal tubular cells. This toxic effect may have a role in the pathophysiology of contrast nephropathy., Methods and Results: We evaluated (i) the cytotoxicity of CM [both low-osmolality (LOCM) and iso-osmolality (IOCM)], of iodine alone, and of an hyperosmolar solution (mannitol 8%) on human embryonic kidney (HEK 293), porcine proximal renal tubular (LLC-PK1), and canine Madin-Darby distal tubular renal (MDCK) cells; and (ii) the effectiveness of various antioxidant compounds [n-acetylcysteine (NAC), ascorbic acid and sodium bicarbonate] in preventing CM cytotoxicity. The cytotoxicity of CM was assessed at different time points, with different methods: cell viability, DNA laddering, flow cytometry, and caspase activation. Both LOCM and IOCM produced a concentration- and time-dependent increase in cell death as assessed by the different methods. On the contrary, iodine alone and hyperosmolar solution did not induce any significant cytotoxic effect. There was not any significant difference in the cytotoxic effect between LOCM and IOCM. Furthermore, both LOCM and IOCM caused a marked increase in caspase-3 and -9 activities and poly(ADP-ribose) fragmentation, while no effect on caspase-8/-10 was observed, thus indicating that the CM activated apoptosis mainly through the intrinsic pathway. Both CM induced an increase in protein expression levels of pro-apoptotic members of the Bcl2 family (Bim and Bad). NAC and ascorbic acid but not sodium bicarbonate had a dose-dependent protective effect on renal cells after 3 h incubation with high dose (200 mg iodine/mL) of both LOCM and IOCM., Conclusion: Both LOCM and IOCM induce a dose-dependent renal cell apoptosis. NAC and ascorbic acid but not sodium bicarbonate prevent this contrast-induced apoptosis.

To define novel pathways that regulate susceptibility to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in non-small cell lung cancer (NSCLC), we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant NSCLC cells, levels of different miRs are increased, and in particular, miR-221 and -222. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. Indeed, transfection with anti-miR-221 and -222 rendered CALU-1-resistant cells sensitive to TRAIL. Conversely, H460-sensitive cells treated with -221 and -222 pre-miRs become resistant to TRAIL. miR-221 and -222 target the 3'-UTR of Kit and p27(kip1) mRNAs, but interfere with TRAIL signaling mainly through p27(kip1). In conclusion, we show that high expression levels of miR-221 and -222 are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets or diagnostic tool for TRAIL resistance in NSCLC.